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RNA-Guided Genome Editing of Mammalian Cells

  • Neena K. Pyzocha
  • F. Ann Ran
  • Patrick D. Hsu
  • Feng Zhang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1114)

Abstract

The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819–823, 2013; Mali P et al., Science 339, 823–826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specific, scalable, and cost-efficient genome editing in mammalian cells.

Key words

CRISPR-Cas Genome editing DNA cleavage Cas9 Guide RNA PAM sequence NHEJ Gene knockout 

Notes

Acknowledgments

We thank Randall Platt for comments and members of the Zhang Lab for discussion, support, and advice. N.P. is supported by the National Science Foundation Graduate Research Fellowship, Primary Award #1122374. P.D.H. is a James Mills Pierce Fellow. F.Z. is supported by the NIH Transformative R01 Award (R01-NS073124); the NIH Director’s Pioneer Award (DP1-MH100706); the Keck, McKnight, Gates, Damon Runyon, Searle Scholars, Merkin, Klingenstein, and Simons Foundations; Bob Metcalfe; Mike Boylan; and Jane Pauley. Sequence and reagent information are available through http://www.genome-engineering.org.

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Neena K. Pyzocha
    • 1
    • 2
  • F. Ann Ran
    • 3
  • Patrick D. Hsu
    • 1
  • Feng Zhang
    • 4
    • 5
    • 6
    • 7
  1. 1.Broad Institute of MIT and Harvard, Massachusetts Institute of TechnologyCambridgeUSA
  2. 2.Department of BiologyMassachusetts Institute of TechnologyCambridgeUSA
  3. 3.Broad Institute of MIT and Harvard, Massachusetts Institute of TechnologyCambridgeUSA
  4. 4.Broad Institute of MIT and HarvardCambridgeUSA
  5. 5.McGovern Institute for Brain ResearchMITCambridgeUSA
  6. 6.Department of Brain and Cognitive SciencesMITCambridgeUSA
  7. 7.Department of Biological EngineeringMITCambridgeUSA

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